The drug discovery process is currently undergoing a fundamental revolution as it embraces `functional genomics`, that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on `positional cloning`. A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position.
Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available. There is a continuing need to identify and characterize further genes and their related polypeptides/proteins, as targets for drug discovery.
It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 1991, 351:353-354). Herein these proteins are referred to as proteins participating in pathways with G-proteins or PPG proteins. Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B. K., et al., Proc. Natl Acad. Sci., USA, 1987, 84:46-50; Kobilka, B. K., et al., Science, 1987, 238:650-656; Bunzow, J. R., et al., Nature, 1988, 336:783-787), G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M. I., et al., Science, 1991, 252:802-8).
For example, in one form of signal transduction, the effect of hormone binding is activation of the enzyme, adenylate cyclase, inside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide, GTP. GTP also influences hormone binding. A G-protein connects the hormone receptor to adenylate cyclase. G-protein was shown to exchange GTP for bound GDP when activated by a hormone receptor. The GTP-carrying form then binds to activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns the G-protein to its basal, inactive form. Thus, the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
The membrane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane .alpha.-helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.
G-protein coupled receptors (otherwise known as 7TM receptors) have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops. The G-protein family of coupled receptors includes dopamine receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders. Other examples of members of this family include, but are not limited to, calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1, rhodopsins, odorant, and cytomegalovirus receptors.
Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure. The 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction.
Phosphorylation and lipidation (palmitylation or famesylation) of cysteine residues can influence signal transduction of some G-protein coupled receptors. Most G-protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus. For several G-protein coupled receptors, such as the .beta.-adrenoreceptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.
For some receptors, the ligand binding sites of G-protein coupled receptors are believed to comprise hydrophilic sockets formed by several G-protein coupled receptor transmembrane domains, said sockets being surrounded by hydrophobic residues of the G-protein coupled receptors. The hydrophilic side of each G-protein coupled receptor transmembrane helix is postulated to face inward and form a polar ligand binding site. TM3 has been implicated in several G-protein coupled receptors as having a ligand binding site, such as the TM3 aspartate residue. TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are also implicated in ligand binding.
G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331). Different G-protein .alpha.-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protein coupled receptors has been identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors. G-protein coupled receptors are found in numerous sites within a mammalian host. Over the past 15 years, nearly 350 therapeutic agents targeting 7 transmembrane (7 TM) receptors have been successfully introduced onto the market.
Polypeptides of the present invention are believed to be members of the Calcitonin/ VIP/Secretin family of 7TM receptors family of polypeptides. They are therefore of interest because calcitonin gene-related peptide (CGRP) receptor belongs to this family of receptors. Calcitonin gene-related peptide (CGRP), amylin and adrenomedullin (ADM) belong to a family of structurally and biologically related polypeptides (Poyner, Trends Pharmacol. Sci., 1995,16: 424-428.)
Calcitonin Gene-Related Peptide is a 37 amino acid polypeptide that is localized at nerve terminals present in the central and peripheral nervous systems (Goodman, Life Sci. 1986,38: 2169-2172) and is an extremely potent vasoactive substance which displays marked chronotropic, inotropic and vasodilatory effects in humans, and animals. (Fischer et al, Bone Miner, 1987,2, 347-359). Amylin is also a 37 amino acid peptide with 47% sequence identity with CGRP and plays a physiological role in regulating glucose metabolism in muscles (Rink et al, Trends Pharmacol. Sci., 1993, 14: 113-118.). Human adrenomedullin (ADM), a 52 amino acid peptide, is a recently discovered vasodilating peptide implicated in the regulation of circulatory homeostasis and pathophysiology of cardiovascular diseases (Samson, Frontiers in Neuroendocrinology 1998,19: 100-127). These bioactive peptides share both the ring structure with a disulfide bridge and the C-terminal amide structure.
Adrenomedullin (ADM) is widely distributed in human tissues, including the adrenal medulla, kidney, and lung, and is found in human plasma, suggesting it may function as a circulating hormone. Studies have demonstrated that ADM is produced and secreted from cultured vascular smooth muscle cells (SMC) and vascular endothelial cells (Schell et al, Trends Endocrinol Melab 1996,7:7-13). When injected intravenously into rats, ADM elicited a strong, long-lasting hypotension, as a result of vasodilation in resistance arteries (Kangawa et al, J. Cardiac Failure 1996,2:S135-S140). Apart from the vasorelaxant effect, ADM has been implicated in the regulation of renal function by its potent natriuretic and diuretic properties, exhibits bronchodilatory effect and inhibits the release of aldosterone and ACTH. ADM has also been shown to down-regulate insulin secretion and blood glucose metabolism (Martinez et al, Endocrinology 1996, 137: 2626-2632). ADM effects are mediated through specific receptors via multiple intracellular transduction pathways (Shimekake et al, J. Biol. Chem. 1995, 270: 4412-4417). Using [.sup.125 I] rat ADM, specific binding sites were demonstrated on various rat tissues. Among the tissues tested, lung had the highest density of ADM binding sites. Studies have shown that ADM stimulates the accumulation of intracellular cAMP in vascular SMC, and bovine EC and induces a rise in intracellular Ca.sup.++ through the activation of phospholipase C (Shimekake et al, J. Biol. Chem. 1995, 270: 4412-4417). ADM induces rapid and transient c-fos gene expression and AP-I DNA binding activity in rat vascular SMC and cardiomyocytes (Sato et al, Biochem. Biophys. Res. Commun. 1995, 217: 211-216). Further, ADM activated the NO-cGMP system in kidney, vascular EC and also in rabbit ventricular myocytes (Ikenouchiet al, Circulation 1997, 95:2318-2324). In normal humans, immunoreactive ADM has been reported to be present in plasma and urine (Kitamuraet al, FEBS Lett. 1994, 341:288-290). In pathological states, the plasma concentration of ADM has been reported to be increased such as in class IV congestive heart failure (5-fold), acute myocardial infarction (5 fold) and the combination of both resulted in increased ADM levels by 10 fold (Massart et al, Acta Cardiol. 196, 51, 259-269). Evidence for cardiac secretion of ADM was also shown to be related to the severity of CHF in patients (Massart et al, Acta Cardiol. 196, 51, 259-269). In addition, the ADM level increased in chronic renal failure (4-fold), hypertension (2-fold), acute asthma attack (5 fold) and sepsis (13-fold). Studies have shown the up-regulation of ADM mRNA in a model of ischemic stroke (Jougasaki et al, J. Clin. Invest. 1996, 97, 2370-2376).
Calcitonin gene-related peptide is an extremely potent vasoactive substance which displays marked chronotropic, inotropic and vasodilatory effects in humans, and animals (Wimalawansa, Endocrine Reviews 1997,17:533-585). The vasodilator effect of CGRP is endothelium dependent or independent, depending on the vascular bed and species involved (Greenberg et al, Br. J. Pharmacol 1987,92, 789, Gray et al, Eur. J Pharmacol 1992, 212:37-42). Infusion of CGRP into humans, and a variety of other species produces vasodilation, increased regional blood flow, hypotension and tachycardia. Studies in rabbit mesenteric resistance arteries and rat basilar artery suggest that CGRP acts through an ATP-sensitive potassium channel to induce vasodilation since in these blood vessels CGRP actions are inhibited by glibenclamide (Nelson et al, Nature 1990, 344: 770-773). In addition to its vasodilatory effects, CGRP enhances the increase in vascular permeability induced by various inflammatory mediators including interleukin, PAF, histamine and bradykinin (McGillis et al, Methods in Neuroscience, 1995, 24: 355-389). CGRP also affects the glomerular filtration rate, renal blood flow, and secretion of renin and has been implicated in pain transmission at sensory spinal pathways in the dorsal horn of the spinal cord. Recent evidence also suggests that CGRP might inhibit the proliferation of smooth muscle cells (Fiscus, FASEB J., 1993, 7, A791. Abstract 4567 ) and lipid peroxidation (Li et al, Med Sci Res., 1995, 23:253-254). CGRP is a potent, indirect antagonist of insulin's effects on glucose metabolism. In "glucose clamp" studies in rats, CGRP was shown to produce "insulin resistance" exemplified by a rapid and sustained reduction in the glucose infusion rate needed to maintain plasma euglycemia during a constant insulin infusion rate (Molina et al, Diabetes 1990, 39: 260-265). Furthermore, CGRP was effective in blocking insulin inhibition of hepatic glucose production. Activation of sensory nerves by capsaicin and resiniferatoxin inhibits insulin-stimulated glycogen synthesis which correlates with an increase of CGRP released from sensory nerves in skeletal muscle. Thus, CGRP has been implicated as a mediator of cerebral vasodilation leading to migraine and may play a role in the onset of Type 11 diabetes (NIDDM) via promotion of insulin resistance.
Calcitonin gene-related peptide initiates its responses through an interaction with specific membrane receptors on target tissue that are primarily coupled to the activation of adenylyl cyclase. CGRP-mediated-activation of adenylyl cyclase have been identified and characterized in several tissues, including cardiac, endothelial and vascular smooth muscle (Poyner, Pharmacol Ther 1992, 56:23-51, Wimalawansa, Endocrine Reviews, 1997, 17:533-585.). The receptor for CGRP has been cloned from various species including human, rat and pig (Aiyar et al, J Biol Chem 1996, 271:11325-11329; Elshourbagy et al, Endocrinology 1998, 139:1678-1683; Han et al, Mol Endocrinology. 1997,18:267-272; Njuki et al, Clinical Sci 1993. 85:385-388). These receptors show 91-95% identity at the amino acid level among species. Related calcitonin receptor like-receptors (CRLR) (Njuki et al, Clinical Sci 1993, 85: 385-388; Fluhmann et al, Biochem Biophys Res Commun 1995, 206:341-347) were reported previously but they were unable to identify the ligand. It is important to note that the expression of functional CGRP receptors could be accomplished only in human embryonic kidney 293 (HEK-293) cells. The CGRP receptor displays 7 transmembrane domains and shows significant homology with a subfamily of G-protein coupled receptors that includes calcitonin, vasoactive intestinal peptide, secretin, glucagon and corticotropin releasing factor (Segreet al, Trends Endocrinol Metab 1993, 4:309-314). Messenger RNA encoding the CGRP receptor is expressed in relatively high levels in human heart and lung. Expression of the recombinant CGRP receptor in stably transfected HEK 293 cells has enabled study of their ligand stimulated signal transduction. Like other receptors of this family, the recombinant CGRP receptor was shown to be capable of activating adenylyl cyclase as well as rapidly increasing intracellular calcium through the activation of phospholipase C (Aiyaret al, Mol Cellular Biochem 1998 (in press)).
Recent evidence (McLatchie et al, Nature 1998, 393, 333-339) suggests that the related peptides, CGRP and ADM, bind to the same seven transmembrane receptor, with receptor specificity being determined by a receptor associated modifying proteins (RAMPs). They demonstrated that when the calcitonin receptor like receptor (CRLR) is coexpressed with RAMP 1, the expressed receptor responds to CGRP. On the other hand, when CRLR is co-expressed with RAMP2, the expressed receptor responds to ADM. Thus, CRLR has been shown to act as both CGRP and ADM receptors which depend upon the cellular expression of specific RAMPs.